The present invention relates to substances which are useful in modifying cellxe2x80x94cell adhesion and in modifying the permeability of physiological barriers.
Cellxe2x80x94cell adhesion is of crucial importance for the development and maintenance of tissue structure. Furthermore, regulation of such adhesion plays a normal role in physiological situations such as tissue turnover. Aberrant control may contribute to the aetiology of pathologies such as cancer and inflammation.
Therefore, there has been considerable interest in the basic processes whereby cells adhere to each other and ways in which such processes may be regulated. In general, cellxe2x80x94cell adhesion involves proteins on neighbouring cells that bind to one another. The cytoplasmic domains of proteins actually involved in adhesion can also physically associate with other cytoplasmic proteins that may either play a mechanical role, such as providing links with other proteins, or provide a regulatory influence.
p120 and p100 are members of the armadillo protein family. The armadillo repeats found in the members of this family appear to provide means whereby proteins can interact with others. p120 and p100, intracellular, cytoplasmic proteins, are now known to associate with cadherins, which are Ca2+-dependent adhesion molecules that contribute in an important way to cellxe2x80x94cell adhesion. Cadherins are important in control of cell proliferation. If cadherins fail to function properly, cells can proliferate in an unregulated fashion and metastasize. Cadherins are also thought to be important components in controlling the permeability of physiological barriers i.e. cell tight junctions. Here, disruption of cadherin based cellxe2x80x94cell adhesion leads to increased tight junction permeability. This has raised the possibility that tight junctions, although potentially regulatable in themselves, may be regulated by the adhesiveness of the adherens junction. In turn, cadherin adhesiveness may be regulated by the phosphorylation state of associated catenins, including p100 and p120.
The blood-brain barrier is an important example of a cell tight junction. It serves to separate the molecular, ionic and cellular environment of the blood from that of the brain. To a major degree, this separation is achieved by inter-endothelial tight junctions of high electrical resistance which greatly diminish paracellular flux. It is clear that the permeability of the tight junctions of the blood-brain barrier is not immutable. Rather, permeability appears to undergo dynamic regulation, but the way in which this is achieved is not fully understood.
In WO95/13820 it is disclosed that tyrosine protein phosphorylation is a key regulator of the permeability of tight junctions in both epithelial and endothelial cells; tyrosine protein phosphorylation may therefore be manipulated to control the permeability of the blood-brain and other physiological barriers. Decreasing the degree of tyrosine protein phosphorylation reduces permeability of the blood-brain or other barrier, whereas increasing the degree of tyrosine protein phosphorylation increases permeability. WO95/13820 also disclosed that the proteins p100 and p120 are believed to be substrates of tyrosine kinase. Further information regarding p100 and p120 is provided in WO96/16170.
Although WO95/13820 and WO96/16170 provide important information regarding the functioning of p100 and p120 this information is not complete. Unexpectedly, the present inventors have now discovered that p100 and/or p120 participate in a cycle which involves the phosphorylation/dephosphorylation of serine/threonine residues on these proteins. Furthermore they have shown that this cycle is regulatable by agents which are known tumour promoters, inflammatory mediators and tight junction permeability modulators. Thus, agents which interfere with regulation of this cycle itself or pathways involved in its regulation could have application to a wide variety of medical situations.
According to the present invention there is provided an agent capable of inducing the phosphorylation of serine and/or threonine residues of p100 and/or p120, for use in medicine.
The present invention also provides an agent capable of inducing the dephosphorylation of phosphorylated serine and/or threonine of p100 and/or p120 residues, for use in medicine.
By using an agent as described above, the permeability of physiological barriers i.e. tight junction permeability could be modified and cellxe2x80x94cell adhesion could also be modified.
By way of example, one way of utilising the present invention is to adjust the activity of protein kinase C. The present inventors have found that dephosphorylation of phosphorylated serine and/or threonine residues in p120 and/or p100 can be induced by increasing protein kinase C activity (i.e. by using protein kinase C activators), and that the phosphorylation of serine and/or threonine residues present in p100 and/or p120 can be induced by decreasing protein kinase C activity (i.e. by using protein kinase C inhibitors).
Examples of protein kinase C inhibitors are phorbol diesters, bryostatins 1 and 2, (xe2x88x92) indolactams V and (+) indolactam V, teleocidind, DHI ([6-(N-Decylamino)-4-hydroxymethylindole]) and ADMB ([3-(N-Acetylamino)-5-(N-decyl-N-methylamino)benzyl alcohol]), lipotoxin A4 and B4, mezerein, (xe2x88x92)-7-octylindolactam V, resiniferatoxin, thymeleatoxin. Protein kinase C may also be activated by ligands that bind to receptors to generate diacylglyccrol. Examples of these are bombesin and other neuropeptides, platelet-derived growth factor, epidermal growth factor.
Examples of protein kinase C inhibitors are A3 ([N-(2-Aminoethyl)-5-chloronaphthalene-1-sulfonamide]xe2x80x94this is optionally combined with HCl), bisindolylmaleimide I (also known as GF 109203X), chelerythrine chloride, Gxc3x66976, Gxc3x67874, H-7 ([1-(5-isoquinolinesulfonyl)-2-methylpiperazine]xe2x80x94this is optionally combined with HCl), hypericin, K-252a, b and c, melittin, phloretin,pseudohypericin, rottlerin, Ro 31-8220, Ro 32-0432, LY333531, (xe2x88x92)balanonl. Other examples are givine in xe2x80x9cDrug Delivery Today, 1996, vol 1, pp438-447xe2x80x9d.
The term xe2x80x9cprotein kinase Cxe2x80x9d, which is sometimes referred to as xe2x80x9cPKCxe2x80x9d, is used herein to refer to a class of enzymes which catalyse the transfer of phosphate from ATP to the serine or threonine residues of polypeptides. Preferably this occurs in a specific manner so that other amino acid residues are not phosphorylated.
Activation of these enzymes can generally be inferred by assaying with MARCKS (a PKC specific substrate). MARCKS is the term used for Myristoylated Alanine Rich CKinase Substrate. This is a protein which was the first major PKC substrate identified. If increased phosphorylation of MARCKS is observed, it can usually be inferred that activation of PKC has occurred.
Inhibitors of PKC can be identified by their ability to prevent or reduce PKC activation.
The present invention is however not limited to the use of activators/inhibitors of PKC since other agents which work independently of PKC but which can affect the level of phosphorylation of threonine and/or serine residues of p100 and/or p120 can be used.
For example, lysophosphatidic acid (LPA) or histamine may be used to induce dephosphorylation of threonine and/or serine residues present in p100 and/or p120 and agents which bind LPA or histamine or blockers of these agents may be used to block the effect of LPA or histamine.
This effect might also be achieved by the use of compositions which are hyperosmolar with respect to the physiological environment proximal to p100 and/or p120 (e.g. hyperosmolar solutions of sugars such as mannitol or arabinose).
Hyperosmotic solutions are believed by some to open up the blood-brain barrier by causing the shrinking of brain endothelial cells which results in the mechanical opening of the endothelial tight junctions. However in view of the information provided herein, it is possible that hyperosmotic treatment triggers intracellular signaling processes leading to p100/p120 dephosphorylation which, as these are junctional proteins, could be responsible for the increase in tight junction permeability.
The present invention is therefore important in indicating that the use of hyperosmolar compositions could be avoided by using other agents to induce dephosphorylation of serine and/or threonine residues present in p100 and/or p120. This is significant since the dangers associated with the use of hyperosmolar solutions can be avoided. (Hyperosmolar solutions such as mannitol solutions are currently used clinically.) These dangers are twofold, firstly there is damage associated with rapid infusion of the hyperosmolar solution via the carotid or vertebral arteries. This includes arterial damage, deep venous thrombosis, pulmonary embolism, granulocytopenia, anaemia, sepsis, hemorrhagic cystitis and interstitial pneumonitis. Secondly, neurologic damage can follow blood-brain barrier opening with hyperosmolar treatment including visual changes, seizures and temporary neurological deterioration.
Other agents useful in the present invention can be identified by screening, as described later.
Particular aspects of the present invention will now be disclosed in greater detail.
Agents of the present invention may be used to modify the permeability of physiological barriers.
a) Increasing Permeability
For example, agents capable of inducing the dephosphorylation of serine and/or threonine residues present in p100 and/or p120 could be used to open physiological barriers temporarily e.g. by increasing the permeability of tight junctions of both endothelial and epithelial cell barriers. These agents are particularly useful in the case of the blood-brain barrier (an example of an endothelial barrier), since they could allow drugs (i.e. therapeutic agents) which would normally not be able to cross this barrier to reach the brain.
The present invention is not however limited to drug delivery to the brain. It could be used for example in the delivery of drugs to other locations e.g. to tumours (especially peripheral tumours) by loosening endothelial cell barriers.
It could also be used, for example, in nasal delivery of drugs by loosening the nasal epithelial cell barrier. Indeed the present invention could be used for providing drug delivery across endothelial and epithelial cell barriers generally.
Where an agent of the present invention is used to facilitate drug delivery it may be administered together with a drug in a single composition.
Alternatively the agent and the drug may be administered separately. Sequential or simultaneous administration of the agent and the drug may be used.
The present invention is not limited to the use of particular drugs since it can be used to deliver a wide variety of drugs to target sites. However, it is anticipated that among the primary candidates for delivery by means of this aspect of the invention will be: anti-tumour compounds, such as methotrexate, adriamycin and cisplatin; growth factors, such as NGF, BDNF and CNTF, which are used to treat neurodegenerative disease; and neurotransmitter antagonists or agonists which do not penetrate the blood-brain barrier (such as certain NMDA receptor blockers).
Another use of the present invention in increasing the permeability of physiological barriers is in opening pulmonary epithelial cell tight junctions. This can act to dilute the accumulation of mucous present in the lungs. It is also useful in aiding the administration of drugs to the lungs, e.g. where an inhaler is used.
b) Decreasing Permeability
Agents capable of inducing the phosphorylation of unphosphorylated serine and/or threonine residues of p100 and/or p120 could be used to decrease the permeability of physiological barriers. This could be achieved for example by decreasing the permeability of tight junctions of both endothelial and epithelial cell barriers. Such agents could be used, for example, in closing tight junctions after they have been opened or in preventing them from opening. In the case of the blood-brain barrier, it will generally be desirable to use these agents to close cell tight junctions in order to avoid possible damage to the brain after they have been opened to allow a drug to cross this barrier.
It is also desirable to close cell tight junctions in the blood-brain barrier for other purposes. For example this can be done:
a) to block or reduce the entry into the brain of lymphocytes which mediate an immune response (useful in treating multiple sclerosis) or of neutrophils which can damage neuronal cells after stroke;
b) to prevent or reduce the entry of metastatic cancer cells into the brain (useful in treating cancer); and
c) to prevent or reduce the risk of brain oedema e.g. following stroke or traumatic head injury (useful in treating head injuries, oedema and stroke).
Cell trafficking across endothelia (such as T cell migration across the blood-brain barrier) and epithelia is believed to occur by several sequential steps. The trafficking cell initially binds to the endothelium or epithelium, at first loosely and then more tightly, migrates to the junctional region and is then believed to migrate through the junction. Clearly, tight junctions must be modulated during this process and it is possible that signalling changes involving p100/p120 serine/threonine phosphorylation may occur and be a necessary part of the transmigration process. Therefore blocking serine/threonine phosphorylation changes in p100 and p120 could block transmigration.
The present invention could also be used to decrease the permeability of other physiological barriers. It could therefore be useful in treating oedema (even if this does not occur in the brain). For example it could be used to treat vasogenic oedema in peripheral tissues by reducing the permeability of the vasculature (e.g. in treating high altitude pulmonary oedema). Reducing the permeability of the vasculature is also useful in preventing or reducing metastases (whether in the brain or elsewhere) by preventing or reducing the ability of tumour cells to exit from the vasculature.
Another use of the present invention in respect of treating tumours is that it could be used to reduce the permeability of leaky junctions of the vasculature supplying solid tumours (i.e. to xe2x80x9ctightenxe2x80x9d these junctions). This can reduce the uptake of nutrients by such cells.
A further use of the present invention is in treating inflammation. This could be achieved by reducing leukocyte migration to peripheral tissues through endothelial cell tight junctions. By reducing the permeability of such junctions leukocyte migration to peripheral tissues can be reduced. The present invention is not however limited to treating inflammation of peripheral tissues. It could be used to treat other inflammatory conditions. For example, it could be used to treat intestinal inflammation. Neutrophil migration across intestinal epithelia can be a cause of intestinal inflammation. By reducing the permeability of epithelial cell junctions intestinal inflammation could therefore be reduced.
A yet further use of the present invention is in treating gastric ulcers. Gastric ulcers can be exacerbated by xe2x80x9cloosexe2x80x9d tight junctions. Thus decreasing the permeability of tight junctions of gastric epithelial cells could be used in the alleviation of gastric ulcers.
From the foregoing passages it is clear that the present invention could be used to treat a wide variety of different disorders by reducing the permeability of cell junctions in order to prevent cells which can cause disorders from reaching sites where such disorders can arise. The present invention could therefore generally be useful in preventing or reducing aberrant cell trafficking.
Agents of the present invention could also be used to modify cellxe2x80x94cell adhesion.
For example, such agents could be used to induce the phosphorylation of serine and/or threonine residues of p120 and/or p100 in order to increase the tendency of cells to adhere to one another.
This could, for example, prevent or slow down the loss of contact inhibition which is seen in the development of cancerous cells. Thus the loss of growth control which is associated with loss of contact inhibition could be prevented or reduced. Such agents could also prevent or slow down the increased cell motility, migration and metastasis, which occurs in the development of cancers.
The agents of the present invention (which could modify the permeability of physiological barriers and which could modify cellxe2x80x94cell adhesion properties) could be used in the preparation of medicaments for use in human or in veterinary medicine.
These medicaments could be used for the treatment of an existing condition or for prophylactic treatment and the word xe2x80x9ctreatmentxe2x80x9d is used herein to cover both of these alternatives.
An agent for use in the present invention may be combined with a pharmaceutically acceptable carrier to form a pharmaceutically acceptable composition. This pharmaceutical composition will usually be sterile. It may be in any suitable form (depending upon the desired method of administering it to a patient). For example, it may be provided in unit dosage or multiple dosage form. It may be provided as a derivative. Thus pharmaceutically acceptable salts, esters or other derivative forms may be used, provided that activity is retained.
Pharmaceutical compositions within the scope of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a carrier under sterile conditions.
Without being bound by theory, a possible scheme is shown in FIG. 1, which will be described later.
In any event the present inventors have established that regulation of the degree of phosphorylation of serine/threonine present in p120/p100 could be used to modify the permeability of physiological barriers and also to modify cellxe2x80x94cell adhesion properties. This finding was not predictable prior to the present invention and is significant in establishing the important regulatory role of p100/p120.
p100 and p120 are described in greater detail in WO95/13820 and WO96/16170, where tyrosine phosphorylation is also discussed. They are proteins with molecular weights of about 100 kDa and about 120 kDa respectively, and are associated with cadherin and cadherin complexes present in endothelial and epithelial cells.
In view of this important finding, the present invention could be used to provide a screen for pharmaceutically active compounds.
For example, p100 and/or p120 could be used to screen for substances capable of increasing the permeability of physiological barriers/reducing cellxe2x80x94cell adhesion. This could be done by screening for substances which can induce the dephosphorylation of phosphorylated threonine and/or serine residues present in p100 and/or p120 (preferably under physiological conditions).
Alternatively p100 and/or p120 could be used to screen for substances capable of decreasing the permeability of physiological barriers/increasing cellxe2x80x94cell adhesion. This could be done by screening for substances which can induce the phosphorylation of threonine and/or serine residues present in p100 and/or p120.
The agents disclosed herein are not limited to being used in medical treatment or in screening since they are generally useful for research into cellxe2x80x94cell adhesion and into physiological barriers (e.g. cell junctions, such as cell tight junctions). In particular, these agents are useful for studying the proteins p100 and p120 and for developing more accurate models of the functions of these proteins. The agents disclosed herein are especially useful in investigating the effects induced by phosphorylation/dephosphorylation of threonine and/or serine residues present in p100 and/or p120.
Agents of the present invention may be used in diagnosis since they can be used to alter the permeability of physiological barriers in order to allow substances which are useful in aiding a diagnosis to reach a desired location. This may be a location which they would not otherwise reach via a particular route of administration. Thus, for example, substances useful in diagnosis which do not normally cross the blood-brain barrier may be allowed to cross this barrier via the present invention. The present invention can also be used to facilitate the entry of diagnostic substances into tumours.
Any suitable diagnostic substances may be used. Antibodies, antibody fragments, lectins or other molecules having high binding specificity may be linked to diagnostic substances in order to target them to a desired site. (Such molecules can also be linked to drugs to target pharmaceutically active substances to a desired site.) For example cancer cells can be targeted.
Preferred diagnostic substances are useful in imaging. For example, they may be useful in providing brain scans.
In the foregoing discussions, references to xe2x80x9cincreasingxe2x80x9d or xe2x80x9cdecreasingxe2x80x9d the permeability of physiological barriers and to xe2x80x9cincreasingxe2x80x9d or xe2x80x9cdecreasingxe2x80x9d cellxe2x80x94cell adhesion are made in the context of administering agents of the present invention to a patient. These shall be taken to include not only absolute increases/decreases in permeability or adhesion but also the increases/decreases in permeability or adhesion relative to the situation which would arise if a patient were left untreated (rather than being treated with an agent for use in the present invention).